Flow cell carrier and methods of use

ABSTRACT

The present disclosure relates to a flow cell carrier. The flow cell carrier may include a flow cell and a frame. The frame may include a pocket and a handle. The pocket may have at least one spring feature and at least one banking feature. The frame may be configured to retain the flow cell within the pocket such that a maximal surface area of the flow cell is exposed to an optical lens. Related methods and kits are also disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/952,787, filed Dec. 23, 2019, which is incorporated herein byreference in its entirety and for all purposes.

BACKGROUND

A tremendous interest in nucleic acid characterization tools was spurredby the mapping and sequencing of the human genome. New tools were neededto cope with the unprecedented amount of genomic information that wasbeing discovered. One such tool that emerged were DNA microarrays; tinygene-based sensors traditionally prepared on coated glass microscopeslides (Southern E., Mir K., and Shchepinov M.; Nature Genetics volume21, p. 5-9 (1999)). A typical DNA microarray included a flat, solidsubstrate (typically glass) with an organic coating, typically anorgano-functional alkoxysilane. The coated glass is grafted with variousknown DNA probes at predefined locations. Standard 25 mm×75 mm glassmicroscope slides were the first supports commonly used for theseinitial microarray assays, which then gave way to the modern flow cell.

Broadly speaking, for nucleic acid sequencing applications, a flow cellmay be considered a reaction chamber that contains one or more nucleicacid templates tethered to a solid support, to which nucleotides andancillary reagents are iteratively applied and washed away. The flowcell allows for imaging of the sites at which the nucleic acids arebound, and resulting image data is used for the desired analysis. Thelatest commercial sequencing instruments use flow cells and massiveparallelization to increase sequencing capacity.

There is a desire to perform high throughput sequencing stemming fromthe need for faster processing and reduced costs. Since the debut of themodern flow cell (Margulies et al; Nature. 2005 Sep 15;437(7057):376-80. 2005), improvements to sequencing flow cells tend tofocus on optimizing spacing patterns and uniform well size as a means toimprove sequencing quality and efficiency. In addition to theseimprovements, there is a general need for a more user-friendly,ergonomically minded, flow cell carrier that reduces costs relative toother known systems and also increases control and efficiency of thereactions intended to be observed. There is, therefore, a continued needfor improved methods and devices for sequencing nucleic acid in order toaddress the practical day-to-day sequencing work of the averagescientist.

BRIEF SUMMARY

In an aspect, a flow cell carrier is provided. The flow cell carrierincludes a flow cell and a frame. The frame includes a handle, and theframe is configured to retain the flow cell.

In another interrelated aspect, a method of manufacturing a flow cellcarrier is provided. The method includes forming a frame such that theframe includes a handle, and securing a flow cell within the frame.

In some variations, one or more of the following features may optionallybe included in any feasible combination.

The frame can further include a pocket, and the pocket can include atleast one biasing feature. The frame can be configured to retain theflow cell within the pocket. The frame can be configured to retain theflow cell within the pocket such that a maximal surface area of the flowcell can be available to be exposed to an optical lens (e.g., theoptical lens of a nucleic acid sequencing device). The optical lens(e.g., the optical lens of the sequencing device) can be configured todetect excitation, emission, or other signals present on the flow cell.The frame can be configured to retain the flow cell within the pocketsuch that a maximal surface area of the flow cell can be available to bein contact with the receiver of a nucleic acid sequencer. The retainingof the flow cell within the pocket further can include constraining afirst, a second, a third, a fourth, a fifth, and a sixth degree offreedom of the flow cell. The frame can be an injection molded frame.The handle can be a raised handle. The frame can be further configuredto provide a gap between a work surface and the flow cell. The framefurther can include at least one ferromagnetic pin. The at least onebiasing feature can be a spring finger. The at least one biasing featurecan be a tab. The flow cell can further include a microchip. Themicrochip can be an electronically erasable programmable read onlymemory (EEPROM) chip.

The forming of the frame can include injection molding. The forming caninclude assembling individual components. The handle can be a raisedhandle. The handle can be an ergonomic handle. The frame can be furtherconfigured to provide a gap between a work surface and the flow cell.The method can further include forming a housing within the frame. Thehousing can be configured to contain a microchip.

In an aspect is provided a method of amplifying and sequencing DNAwithin a flow cell. The method includes providing a flow cell carrier asdescribed herein and positioning the flow cell carrier within a nucleicacid sequencing device. The method also includes flowing a first aqueoussolution that includes one or more reagents and enzymes for DNAamplification through the flow channel. In embodiments, the methodincludes one or more wash cycles. In embodiments, the method includesflowing a sequencing solution through the flow channel that includes oneor more reagents and enzymes for DNA sequencing.

In another aspect is provided a kit, wherein the kit includes a flowcell carrier as described herein. In embodiments, the flow cell carrierincludes a flow cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a top perspective view of an embodiment of a flow cellcarrier consistent with implementations of the current subject matter.

FIG. 1B is a bottom perspective view of the flow cell carrier of FIG.1A.

FIG. 1C is a side view of the flow cell carrier of FIG. 1A.

FIG. 2 is a bottom perspective view of another embodiment of a flow cellcarrier consistent with implementations of the current subject matter.

FIG. 3 is a block diagram of another embodiment of a flow cellconsistent with implementations of the current subject matter.

FIG. 4A is a top perspective view of another embodiment of a flow cellcarrier consistent with implementations of the current subject matter.

FIG. 4B is a bottom perspective view of the flow cell carrier of FIG.4A.

DETAILED DESCRIPTION

Grasping and lifting a flat object from a surface (e.g., a microscopeslide on a table top) is an ongoing research topic, especially in thefield of robotics. Robotics and automation are currently used in thefield of life sciences to aid in performing large-scale and/orhigh-throughput sequencing of proteins, DNA, and the like. Even forhumans with ample dexterity and control, manipulating traditionalsequencing flow cells, i.e. a glass slide containing small fluidicchannels, is challenging and if not performed properly, expensivemishaps can occur. Incorporating an ergonomically designed frame, whichincludes a raised handle, allows for greater access and enables the userto manipulate the flow cell without the risk of dropping or breaking thesensitive reaction chamber.

Disclosed herein is a flow cell carrier that is configured to beergonomically used to grasp, lift, or otherwise support a flow cell. Inan aspect is provided a flow cell carrier including a frame thatincludes a handle; wherein the frame is configured to retain a flowcell. The flow cell carrier has a support platform that supports theflow cell and a handle that can be grasped by a user to lift andtransport a flow cell mounted on the support platform. The supportplatform lifts the flow cell above a work surface and reduces possiblecontamination to the flow cell. The handle significantly reduces therisk of damaging the flow cell by squeezing, scratching, dropping, orotherwise disfiguring the flow cell by hand. Additionally, because flowcells are often used in repetitive tasks, the ergonomic handle reducesrisk of injury to a user from prolonged repetitive motion. Workflowefficiency can also be improved when using the flow cell carrier, as theflow cell can be transported quickly and safely between surfaces withminimal damage to the flow cell. In embodiments, the flow cell carrieris configured to removably engage within a nucleic acid sequencingdevice. The term “removably engage” describe a relationship between theflow cell carrier and a receiving unit of a bioanalytical device, orinterface of a bioanalytical device (e.g., platen receiver of a nucleicacid sequencing system), and is intended to mean that a connectionbetween the flow cell carrier and the receiving unit of a bioanalyticaldevice is readily separable without destroying the receiving unit of abioanalytical device.

FIGS. 1A-1C illustrate an embodiment of a flow cell carrier 100. Theflow cell carrier 100 can include or support a flow cell 110 and a frame150, the frame 150 including a pocket 105 configured to retain the flowcell 110. In embodiments, the frame 150 can further include an appendageor handle 120. For example, the handle 120 may have an ergonomic shape.An ergonomic shape can act to alleviate or prevent pain, or otherundesirable effects, associated with the repetitive hand motionsinvolved in the daily sequencing work of a scientist. An ergonomic shapecan also allow for greater digit (i.e. finger) access and enables theuser to manipulate the flow cell without the risk of damage orcontamination. The pocket 105 acts as a platform or other structure thatsupports and aids in retaining the flow cell 110 in place, such as fortransport. In this regard, the pocket 105 can be a platform sized andshaped to support the flow cell 110 in a horizontal orientation. Thehandle 120 extends from the pocket 105, such as in an upward and outwarddirection and includes a portion that can be grasped by a user, such asby a single hand of the user. In embodiments, the flow cell carrier 100includes an identifying feature such as a label, logo, lot number, or aunique identifying number.

The handle may include an identifying feature, such as a label, logo,lot number, or a unique identifying number. It should be appreciatedthat the shape of the flow cell carrier 100 shown in FIGS. 1A-1C is anexample and that the shape can vary. The handle can be of varyingwidths, lengths, and shapes. In one embodiment, the elongated handle isan ergonomically designed structure to fit comfortably in a user's hand.The handle can be made of various materials known in the art. Inembodiments, the handle is injection molded or assembled by hand andpermanently affixed to the frame. The handle can be provided in varioussizes, including small, medium, and large sizes to accommodate all handsizes, including male, female, adult, adolescent, and small hand sizes.The handle can be of various colors and aesthetic designs. In certainembodiments, the handle can have finger grooves, and/or grippingmaterials deposited thereon.

The frame 150 can include or be coupled to one or more retaining orpositioning components that are configured to retain the flow cell 110in place when the flow cell 110 is positioned in the pocket 105 when ona platen receiver of a bioanalytical instrument (e.g., a nucleic acidsequencer platen). The retaining components are configured to retainand/or position the flow cell 110 in place or otherwise reduce thelikelihood that the flow cell 110 will disengage from the pocket 105when on a platen receiver of a bioanalytical instrument. For example,the frame 150 can be configured to retain the flow cell 110 within thepocket 105 such that a maximal surface area of the flow cell 110 is incontact with the pocket 105 on a platen receiver of a bioanalyticalinstrument. Some examples of such retaining or positioning componentsinclude pins, hooks, posts, or clips. In embodiments, the frame 150 canfurther include a proximal ferromagnetic pin 130 a and a distalferromagnetic pin 130 b. In embodiments, the ferromagnetic pin includesiron, cobalt, or nickel, or oxides thereof. In embodiments, theferromagnetic pin is an alloy containing a transition metal (e.g., Fe,Co, or Ni) and a metalloid (e.g., B, C, Si, P or Al). In embodiments,the frame 150 can further include a proximal magnetic pin 130 a and adistal magnetic pin 130 b. In embodiments, the frame 150 can furtherinclude a proximal steel pin 130 a and a distal steel pin 130 b. In someembodiments, the proximal steel pin 130 a and the distal steel pin 130 bfurther comprise a nickel coating or nickel alloy coating. The pins 130can be located at opposite ends of the pocket 105. For example, the pin130 a is located at or near the handle 150 while the pin 130 b islocated at an opposite end of the pocket 105. The positions, size, shapeand material of the pins can vary.

In example embodiments, the frame 150 can further include a proximalspring finger 140 a positioned along a first length or short edge of thepocket 105. For example, the short edge of the pocket 105 can beperpendicular to a second length or long edge of the pocket 105. Adistal spring finger 140 b and a distalmost spring finger 140 c can bepositioned along the long edge of the pocket 105. In embodiments, theproximal spring finger 140 a, the distal spring finger 140 b, and thedistalmost spring finger 140 c can be molded into the frame 150 topositively locate the flow cell 110 in the pocket 105. In theillustrated embodiment, the spring fingers 140 comprise structures thatextend outwardly from the frame toward the pocket 105. The springfingers engage, abut, or otherwise press against a flow cell 110 mountedon the pocket 105 such as to retain the flow cell in place or to assistin properly positioning the flow cell. It should be appreciated thestructure of the spring fingers can vary and can be any type of springor biasing member that exerts a force onto the flow cell 110 to retainor position the flow cell.

For example, the flow cell 110 can be positively located on the frame bybiasing the flow cell 110 toward a proximal banking tab 160 a and adistal banking tab 160 b positioned along a long edge of the pocket 105opposite the long edge of the pocket 105 containing the distal springfinger 140 b and the distalmost spring finger 140 c, as depicted in FIG.1B. The flow cell 110 is biased toward a distalmost banking tab 160 cpositioned along a short edge of the pocket 105 opposite the short edgecontaining the proximal spring finger 140 a

The pocket 105 defines multiple contact points (such as three contactpoints) for the plane of the flow cell 110. The pocket can constrain theflow cell 110 in multiple degrees of freedom, such as six degrees offreedom. Constraining is used in accordance with its ordinary meaning inthe art and refers to partially restricted movement or completeimmobilization. Six degrees of freedom refers to the freedom of movementof the flow cell 110 in a three-dimensional space. Specifically, theflow cell 110 is free to move along and rotate around threeperpendicular X-, Y-, and Z-axes. A first degree of freedom can bedefined as moving left and right along the X-axis. A second degree offreedom can be defined as moving backward and forward along the Y-axis.A third degree of freedom can be defined as moving up and down along theZ-axis. A fourth degree of freedom can be defined as rotating around theX-axis, or “roll.” A fifth degree of freedom can be defined as rotatingaround the Y-axis, or “pitch.” A sixth degree of freedom can be definedas rotating around the Z-axis, or “yaw.” The pocket 105 or othercomponent of the frame can constrain the flow cell 110 in one or more ofthese degrees of freedom. The purpose of the pocket 105 plane and theproximal spring finger 140 a and distal spring finger 140 b is to fullyconstrain the flow cell 110 in all six degrees of freedom.

FIG. 1B is a bottom perspective view of the flow cell carrier 100 ofFIG. 1A. As described above and shown in FIG. 1B, the proximal bankingtab 160 a and the distal banking tab 160 b are located along a long edgeof the pocket 105, and a distalmost banking tab 160 c is located along ashort edge of the pocket 105. In embodiments, the flow cell carrier 100can also include an electronically erasable programmable read onlymemory (EEPROM) chip 170. For example, the EEPROM chip 170 can beaffixed in a pocket, other structure molded into the frame 150, ordirectly to the frame 150 itself In embodiments, the EEPROM chip 170 canserve two functions; first, to store identifying information (e.g., lotnumbers, sample run, etc.), and second, to provide positive feedbackthat the flow cell 110 has been inserted correctly into the pocket 105.This is in contrast to alternative sequencing flow cell cartridges,which typically employ a single radio frequency identification (RFID)tag (Eltoukhy et al.). The flow cell carrier 100 described herein,including embodiments, can be configured such that the EEPROM chip 170cannot make contact with electronic components of another device orinstrument unless the flow cell 110 is fully seated in the device in aproper location and orientation. Optionally, the flow cell carrier 100can include an EEPROM chip 170 and a RFID tag. Also shown in FIG. 1B arethe proximal steel pin 130 a and the distal steel pin 130 b.

FIG. 1C is a side view of the flow cell carrier 100, showing the frame150 with the handle 120, and the proximal steel pin 130 a and the distalsteel pin 130 b. As shown in FIG. 1C, in embodiments the frame 150 canprovide a gap between the flow cell 110 and a surface on which the flowcell carrier 100 sits. By including a gap between the flow cell 110 andthe surface, it is possible to mitigate damage to the flow cell 110,reduce potential contamination of the flow cell 110, and enable moreefficient processing due to the ease with which the flow cell 110 can beremoved from the surface. In embodiments, the gap can be about 1 mm toabout 10 mm. In embodiments, the gap is about 2.5 mm. In embodiments,the gap is about 5 mm. In embodiments, the gap is about 7.5 mm. Inembodiments, when the flow cell carrier is positioned on thebioanalytical device receiver (e.g., the platen of a nucleic acidsequencing instrument), the receiver complements the flow cell carriersuch that the gap is reduced, minimized, or eliminated and the flow cellis in direct contact with the platen. In embodiments, the flow cell isin direct contact with the receiver of the sequencing device. Thereceiver may include a thermal element, which is configured to transferor absorb thermal energy of the fluid in the flow cell channel.

FIG. 2 is a bottom perspective view of another embodiment of a flow cellcarrier 200. The flow cell carrier 200 can include or support a flowcell 210 and a frame 250, the frame 250 including a pocket configured toretain the flow cell 210. For example, the flow cell 210 can bepositively located on the frame 250 by biasing the flow cell 210 towardthe frame 250 via a proximal biasing feature positioned along a shortedge of the pocket. For example, depicted in FIG. 2, the biasingfeatures are underneath the tabs 240 a and 240 b. The tabs 240 a and 240b may be made of any appropriate material, such as plastic or metal. Insome embodiments, the proximal biasing feature underneath 240 a and thedistal biasing feature underneath 240 b comprise plates, springs, ortabs. For example, the proximal biasing feature underneath 240 a and thedistal biasing feature underneath 240 b can comprise a metal or rigidpolymer. In embodiments, the tabs 240 a and 240 b can have a thicknessin the range of about 0.05 mm to about 0.5 mm. In embodiments, the tabs240 a and 240 b can have a thickness of about 0.1 mm. In embodiments,the tabs 240 a and 240 b can have a thickness of about 0.25 mm. The tabs240 a and 240 b can be affixed to the frame 250 via heat staking,ultrasonic peening, or other suitable methods. Additionally, the flowcell carrier 200 can include a retaining feature 260, such as a wedge orblock, configured to retain the tab 240 a. The retaining feature 260 maybe removable, or may be permanently affixed to the frame 250. The flowcell may be removed from the flow cell carrier, for example when thewedge 260 is present the flow cell (e.g., the glass slide containing oneor more channels) may be removed and a new flow cell may be insertedinto the carrier. Alternatively, the flow cell may not be removed fromthe flow cell carrier, for example heat staking the tabs 240 a and 240 bprevents reuse of the flow cell carrier. In embodiments, the frame 250can further include an appendage or handle 220 and/or an EEPROM chip270.

FIG. 3 is a block diagram of an embodiment of a flow cell 310. The flowcell 310 can include a substrate 315 having at least one channel 325.The flow cell 310 can be used in a flow cytometer or similar instrument,and may be used for cell counting and/or analysis, spectroscopy,immunoassays, genetic sequencing, DNA sequencing, protein sequencing,scattering, particle counting, flow visualization, or the like. Inembodiments, the flow cell 310 can be used in a nucleic acid sequencingsystem (or nucleic acid sequencer) configured for various applications,including de novo sequencing, sequencing of whole genomes, or targetgenomic regions, or metagenomics. In embodiments, the at least onechannel 325 can be a microfluidic channel configured to allow a materialto flow therethrough. The flow cell may include a plurality of inletports sized to receive a flow of reagent into the flow cell and aplurality of outlet ports that pass the flow of reagent out of the flowcell. A flow channel of the flow cell is positioned between, and influid communication with, each inlet port and the outlet port. The terms“fluid communication” or “fluidically coupled” refers to two spatialregions being connected together such that a liquid or gas may flowbetween the two spatial regions. In embodiments, the at least onechannel 325 can be a reaction channel configured to subject a materialwithin the at least one channel 325 to a desired chemical or physicalreaction. In embodiments, the at least one channel 325 is avisualization channel configured to observe or record physical orchemical properties of a material within the at least one channel 325.For example, the at least one channel 325 can be formed on the substrate315 by etching, engraving, cutting, or other similar means suited to thematerials used in the substrate 315 and the desired characteristics ofthe at least one channel 325. In embodiments, each of the at least onechannel 325 can have an input 335 and an output 345. For example, theinput 335 can be configured to allow a stream of cells to enter the atleast one channel 325, flow through the at least one channel 325, andexit the at least one channel 325 via the at least one output 345.

FIGS. 4A-4B illustrate another embodiment of a flow cell carrier 400.The flow cell carrier 400 can include or support at least one flow cell410 and a frame 450, the frame 450 including a pocket 405 configured toretain the flow cell 410. In embodiments, the frame 450 can furtherinclude an appendage or handle 420. For example, the handle 420 may havean ergonomic shape and design elements as described above. The handle420 may include ridges, as shown in FIG. 4A, or other texture such asgrooves, indentations, rippling, stippling, or the like, to improvegrip. The pocket 405 acts as a platform or other structure that supportsand aids in retaining the flow cell 410 in place, such as for transport.In this regard, the pocket 405 can be a platform sized and shaped tosupport the flow cell 410 in a horizontal orientation. The handle 420extends from the pocket 405, such as in an upward and outward directionand includes a portion that can be grasped by a user, such as by asingle hand of the user. In embodiments, the flow cell carrier 400includes an identifying feature such as a label, logo, lot number, or aunique identifying number. The handle may include an identifyingfeature, such as a label, logo, lot number, or a unique identifyingnumber. It should be appreciated that the shape of the flow cell carrier400 shown in FIGS. 4A-4B is an example and that the shape can vary.

The frame 450 can include or be coupled to one or more retaining orpositioning components that are configured to retain the flow cell 410in place when the flow cell 410 is positioned in the pocket 405 when ona platen receiver of a bioanalytical instrument (e.g., a nucleic acidsequencer platen). The retaining components are configured to retainand/or position the flow cell 410 in place or otherwise reduce thelikelihood that the flow cell 410 will disengage from the pocket 405.For example, the frame 450 can be configured to retain the flow cell 410within the pocket 405 such that a maximal surface area of the flow cell410 is in contact with the platen receiver of a bioanalytical instrument(e.g., a nucleic acid sequencer platen. In embodiments, the frame 450can further include a proximal ferromagnetic pin 430 a and a distalferromagnetic pin 430 b. In embodiments, the ferromagnetic pin includesiron, cobalt, or nickel, or oxides thereof. In embodiments, theferromagnetic pin is an alloy containing a transition metal (e.g., Fe,Co, or Ni) and a metalloid (e.g., B, C, Si, P or Al). In embodiments,the frame 450 can further include a proximal magnetic pin 430 a and adistal magnetic pin 430 b. In embodiments, the frame 450 can furtherinclude a proximal steel pin 430 a and a distal steel pin 430 b. In someembodiments, the proximal steel pin 430 a and the distal steel pin 430 bfurther comprise a nickel coating or nickel alloy coating. The pins 430a and 430 b can be located at opposite ends of the pocket 405. Forexample, the pin 430 a is located at or near the handle 450 while thepin 430 b is located at an opposite end of the pocket 405. Thepositions, size, shape and material of the pins can vary.

In example embodiments, the flow cell 410 can be positively located onthe frame 450 by biasing the flow cell 410 toward the frame 450 via aproximal biasing feature positioned along a short edge of the pocket405. For example, depicted in FIG. 4B, the biasing features areunderneath the tabs 440 a and 440 b. The biasing features aid inpositioning the flow cell in the X and Y directions. The tabs 440 a and440 b may be made of any appropriate material, such as plastic or metal.In some embodiments, the proximal biasing feature underneath tab 440 aand the distal biasing feature underneath tab 440 b comprise plates,springs, or tabs. For example, there may be a proximal bump in themolded plastic frame 450 and a distal metal leaf spring under tab 440 bto create a Y-direction bias. The proximal biasing feature underneathtab 440 a and the distal biasing feature underneath tab 440 b cancomprise a metal or rigid polymer. In embodiments, the tabs 440 a and440 b can have a thickness in the range of about 0.05 mm to about 0.5mm. In embodiments, the tabs 440 a and 440 b can have a thickness ofabout 0.1 mm. In embodiments, the tabs 440 a and 440 b can have athickness of about 0.25 mm. The tabs 440 a and 440 b can be affixed tothe frame 450 via heat staking, ultrasonic peening, or other suitablemethods. Additionally, the flow cell carrier 400 can include a retainingfeature 460, such as a wedge or block, configured to retain the tab 440a. The retaining feature 460 may be removable, or may be permanentlyaffixed to the frame 450. In embodiments, the frame 450 can furtherinclude an appendage or handle 420 and/or an EEPROM chip 470. Inembodiments, such as that shown in FIG. 4B, the EEPROM chip 470 may berecessed in the frame 450.

FIG. 4B is a bottom perspective view of the flow cell carrier 400 ofFIG. 4A. In embodiments, the flow cell carrier 400 can also include anelectronically erasable programmable read only memory (EEPROM) chip 470.For example, the EEPROM chip 470 can be affixed in a pocket 475, otherstructure molded into the frame 450, or recessed directly to the frame450 itself In embodiments, the EEPROM chip 470 can serve at least twofunctions; first, to store identifying information (e.g., lot numbers,sample run, date of manufacture, etc.), and second, to provide positivefeedback that the flow cell 410 has been inserted correctly into thepocket 405. Additionally, the EEPROM chip 470 can provide instructionsor protocols to the sequencing device. This is in contrast toalternative sequencing flow cell cartridges, which typically employ asingle radio frequency identification (RFID) tag (Eltoukhy et al.), andavoids the use of antennas, receivers, or additional power sources . Theflow cell carrier 400 described herein, including embodiments, can beconfigured such that the EEPROM chip 470 (alternatively referred to as170 in FIG. 1B or 270 in FIG. 2) cannot make contact with electroniccomponents of another device or instrument unless the flow cell 410 isfully seated in the device in a proper location and orientation.Optionally, the flow cell carrier 400 can include both an EEPROM chip470 and a RFID tag. Also shown in FIG. 4B are the proximal steel pin 430a and the distal steel pin 430 b.

In an aspect is provided a method of amplifying and sequencing DNAwithin a flow cell. The method includes providing a flow cell carrier asdescribed herein and positioning the flow cell carrier within a nucleicacid sequencing device. In embodiments, the method of sequencing anucleic acid includes providing a flow cell carrier as described herein,wherein the flow cell is configured to serve as a reaction vessel in anucleic acid sequencing device; positioning the flow cell carrier on thereceiver of a nucleic acid sequencing device wherein the flow cell isexposed to an optical lens; and conducting a plurality of sequencingevents and detecting the sequencing events (e.g., a sequencing event asdescribed herein) using the optical lens, thereby sequencing a nucleicacid.

In embodiments, the method includes amplification methodologiesdescribed herein or known in the art. Suitable methods for amplificationinclude, but are not limited to, the polymerase chain reaction (PCR),strand displacement amplification (SDA), transcription mediatedamplification (TMA) and nucleic acid sequence based amplification(NASBA), for example, as described in U.S. Pat. No. 8,003,354, which isincorporated herein by reference in its entirety. In embodiments,amplifying includes thermal bridge polymerase chain reactionamplification; for example, as exemplified by the disclosures of U.S.Pat. Nos. 5,641,658; 7,115,400; 7,790,418; U.S. 2008/0009420, each ofwhich is incorporated herein by reference in its entirety. The aboveamplification methods can be employed to amplify one or more nucleicacids of interest within a flow cell. For example, PCR, multiplex PCR,SDA, TMA, NASBA and the like can be utilized to amplify immobilizednucleic acid fragments within a flow cell.

A nucleic acid can be amplified by a thermocycling method or by anisothermal amplification method. In some embodiments a rolling circleamplification method is used. In some embodiments, amplification takesplace on a solid support (e.g., within a flow cell) where a nucleicacid, nucleic acid library or portion thereof is immobilized. In certainsequencing methods, a nucleic acid library is added to a flow cell andimmobilized by hybridization to anchors under suitable conditions. Thistype of nucleic acid amplification is often referred to as solid phaseamplification. In some embodiments of solid phase amplification, all ora portion of the amplified products are synthesized by an extensioninitiating from an immobilized primer. Solid phase amplificationreactions are analogous to standard solution phase amplifications exceptthat at least one of the amplification oligonucleotides (e.g., primers)is immobilized on a solid support.

Nucleic acid sequencing systems utilize excitation beams to excitelabeled nucleotides in the DNA containing sample to enable analysis ofthe base pairs present within the DNA. Many of the next-generationsequencing (NGS) technologies use a form of sequencing by synthesis(SBS), wherein modified nucleotides are used along with an enzyme toread the sequence of DNA templates in a controlled manner. Inembodiments, sequencing includes a sequencing by synthesis event, whereindividual nucleotides are identified iteratively (e.g., incorporatedand detected into a growing complementary strand), as they arepolymerized to form a growing complementary strand. In embodiments,nucleotides added to a growing complementary strand include both a labeland a reversible chain terminator that prevents further extension, suchthat the nucleotide may be identified by the label before removing theterminator to add and identify a further nucleotide. Such reversiblechain terminators include removable 3′ blocking groups, for example asdescribed in U.S. Pat. Nos. 10,738,072, 7,541,444 and 7,057,026. Oncesuch a modified nucleotide has been incorporated into the growingpolynucleotide chain complementary to the region of the template beingsequenced, there is no free 3′-OH group available to direct furthersequence extension and therefore the polymerase cannot add furthernucleotides. Once the identity of the base incorporated into the growingchain has been determined, the 3′ reversible terminator may be removedto allow addition of the next successive nucleotide. In embodiments, thenucleic acid sequencing system utilizes the detection of four differentnucleotides that comprise four different labels.

I. Definitions

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby expressly incorporated hereinby reference in their entireties.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the disclosure, some preferred methods and materials aredescribed. Accordingly, the terms defined immediately below are morefully described by reference to the specification as a whole. It is tobe understood that this disclosure is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context in which they are used by those of skill inthe art.

As used herein, the singular terms “a”, “an”, and “the” include theplural reference unless the context clearly indicates otherwise.

Reference throughout this specification to, for example, “oneembodiment”, “an embodiment”, “another embodiment”, “a particularembodiment”, “a related embodiment”, “a certain embodiment”, “anadditional embodiment”, or “a further embodiment” or combinationsthereof means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of ” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

As used herein, the term “nucleic acid” refers to nucleotides (e.g.,deoxyribonucleotides or ribonucleotides) and polymers thereof in eithersingle-, double- or multiple-stranded form, or complements thereof. Theterms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, inthe usual and customary sense, to a sequence of nucleotides. The term“nucleotide” refers, in the usual and customary sense, to a single unitof a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA, and hybridmolecules having mixtures of single and double stranded DNA and RNA withlinear or circular framework. Non-limiting examples of polynucleotidesinclude a gene, a gene fragment, an exon, an intron, intergenic DNA(including, without limitation, heterochromatic DNA), messenger RNA(mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinantpolynucleotide, a branched polynucleotide, a plasmid, a vector, isolatedDNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, anda primer. Polynucleotides useful in the methods of the disclosure maycomprise natural nucleic acid sequences and variants thereof, artificialnucleic acid sequences, or a combination of such sequences.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

As used herein, the term “polynucleotide template” refers to anypolynucleotide molecule that may be bound by a polymerase and utilizedas a template for nucleic acid synthesis. As used herein, the term“polynucleotide primer” refers to any polynucleotide molecule that mayhybridize to a polynucleotide template, be bound by a polymerase, and beextended in a template-directed process for nucleic acid synthesis, suchas in a PCR or sequencing reaction. Polynucleotide primers attached to acore polymer within a core are referred to as “core polynucleotideprimers.”

In general, the term “target polynucleotide” refers to a nucleic acidmolecule or polynucleotide in a starting population of nucleic acidmolecules having a target sequence whose presence, amount, and/ornucleotide sequence, or changes in one or more of these, are desired tobe determined. In general, the term “target sequence” refers to anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,cDNA, RNA including mRNA, miRNA, rRNA, or others. The target sequencemay be a target sequence from a sample or a secondary target such as aproduct of an amplification reaction. A target polynucleotide is notnecessarily any single molecule or sequence. For example, a targetpolynucleotide may be any one of a plurality of target polynucleotidesin a reaction, or all polynucleotides in a given reaction, depending onthe reaction conditions. For example, in a nucleic acid amplificationreaction with random primers, all polynucleotides in a reaction may beamplified. As a further example, a collection of targets may besimultaneously assayed using polynucleotide primers directed to aplurality of targets in a single reaction. As yet another example, allor a subset of polynucleotides in a sample may be modified by theaddition of a primer-binding sequence (such as by the ligation ofadapters containing the primer binding sequence), rendering eachmodified polynucleotide a target polynucleotide in a reaction with thecorresponding primer polynucleotide(s).

As used herein, the term “flow cell” refers to the reaction vessel in anucleic acid sequencing device. The flow cell is typically a glass slidecontaining small fluidic channels (e.g., a glass slide 75 mm×25 mm×1 mmhaving one or more channels), through which sequencing solutions (e.g.,polymerases, nucleotides, and buffers) may traverse. Though typicallyglass, suitable flow cell materials may include polymeric materials,plastics, silicon, quartz (fused silica), Borofloat® glass, silica,silica-based materials, carbon, metals, an optical fiber or opticalfiber bundles, sapphire, or plastic materials such as COCs and epoxies.The particular material can be selected based on properties desired fora particular use. For example, materials that are transparent to adesired wavelength of radiation are useful for analytical techniquesthat will utilize radiation of the desired wavelength. Conversely, itmay be desirable to select a material that does not pass radiation of acertain wavelength (e.g., being opaque, absorptive, or reflective). Inembodiments, the material of the flow cell is selected due to theability to conduct thermal energy. In embodiments, a flow cell includesinlet and outlet ports and a flow channel extending therebetween.

Several examples herein are demonstrated for a rectangular flow cellhaving elongated channels. The flow cell is not intended to be limitedto any particular size, though typical flow cells are about 75 mm×25 mm.The depth (i.e., the thickness) of the flow cell depends on theparticular use, for example the flow cell may be about 75 mm×25mm×0.5-2.0 mm. In embodiments, the flow cell is capable of being removedfrom the flow cell carrier. In embodiments, the flow cell is permanentlyaffixed to the flow cell carrier. Flow cells may have one or morefluidic channels in which a polynucleotide is displayed (e.g., whereinpolynucleotides are directly attached to the flow cell or wherein thepolynucleotides are attached to one or more beads arrayed upon or withina flow cell channel) and can be comprised of glass, silicon, plastic, orvarious combinations thereof. In embodiments, the flow cell can includedifferent numbers of channels (e.g., 1 channel, 2 or more channels, 4 ormore channels, or 6, 8, 10, 16 or more channels, etc.). Additionally,the flow cell can include channels of different depths and/or widths(different both between channels in different flowcells and differentbetween channels within the same flow cell). For example, while thechannels may be 50 μm deep, 100 μm deep, or 500 μm deep. Flow cellstypically hold a sample (e.g., a plurality of nucleic acid clusters)along a surface for imaging by an external imaging system. Flow cellsprovide a convenient format for housing an array of nucleic acids thatis subjected to a sequencing-by-synthesis (SBS) or other sequencingtechnique that involves repeated delivery of reagents in cycles.Examples of flowcells and related fluidic systems and detectionplatforms that can be readily used in the methods of the presentdisclosure are described, for example, in Bentley et al., Nature456:53-59 (2008). Alternatively, in embodiments, the flow cell includesa plurality of open wells (e.g., wells of a multi-well plate, surface ofa chip, or surface of a sheet).

In embodiments, the flow cell includes one or more channels each havingat least one transparent window. In particular embodiments, the windowcan be transparent to radiation in a particular spectral rangeincluding, but not limited to x-ray, ultraviolet (UV), visible (VIS),infrared (IR), microwave and/or radio wave radiation. In embodiments,one or more windows can provide a view to an internal substrate to whichpolynucleotides are attached. Exemplary flow cells and physical featuresof flow cells that can be useful in a method or apparatus set forthherein are described, for example, in US 2010/0111768, US 2011/0059865or US 2012/0270305, each of which is incorporated herein by reference inits entirety.

As used herein, the term “channel” refers to a passage in or on asubstrate material that directs the flow of a fluid. A channel may runalong the surface of a substrate, or may run through the substratebetween openings in the substrate. A channel can have a cross sectionthat is partially or fully surrounded by substrate material (e.g., afluid impermeable substrate material). For example, a partiallysurrounded cross section can be a groove, trough, furrow or gutter thatinhibits lateral flow of a fluid. The transverse cross section of anopen channel can be, for example, U-shaped, V-shaped, curved, angular,polygonal, or hyperbolic. A channel can have a fully surrounded crosssection such as a tunnel, tube, or pipe. A fully surrounded channel canhave a rounded, circular, elliptical, square, rectangular, or polygonalcross section. In particular embodiments, a channel can be located in aflow cell, for example, being embedded within the flow cell. A channelin a flow cell can include one or more windows that are transparent tolight in a particular region of the wavelength spectrum. In embodiments,the channel contains one or more polymers of the disclosure. Inembodiments, the channel is filled by the one or more polymers, and flowthrough the channel (e.g., as in a sample fluid) is directed through thepolymer in the channel. In embodiments, the channel contains a gel. Theterm “gel” in this context refers to a semi-rigid solid that ispermeable to liquids and gases. Exemplary gels include, but are notlimited to, those having a colloidal structure, such as agarose; polymermesh structure, such as gelatin; or cross-linked polymer structure, suchas polyacrylamide or a derivative thereof. Analytes, such aspolynucleotides, can be attached to a gel or polymer material viacovalent or non-covalent means. Exemplary methods and reactants forattaching nucleic acids to gels are described, for example, in US2011/0059865 which is incorporated herein by reference. The analytes canbe nucleic acids and the nucleic acids can be attached to the gel orpolymer via their 3′ oxygen, 5′ oxygen, or at other locations alongtheir length such as via a base moiety of the 3′ terminal nucleotide, abase moiety of the 5′ nucleotide, and/or one or more base moietieselsewhere in the molecule. In embodiments, the shape of the channel caninclude sides that are curved, linear, angled or a combination thereof.Other channel features can be linear, serpentine, rectangular, square,triangular, circular, oval, hyperbolic, or a combination thereof. Thechannels can have one or more branches or corners. The channels canconnect two points on a substrate, one or both of which can be the edgeof the substrate. The channels can be formed in the substrate materialby any suitable method. For example, channels can be drilled, etched, ormilled into the substrate material. In embodiments, the channels of theflow cell can be formed in the substrate material prior to bondingmultiple layers together. Alternatively, or additionally, the channelscan be formed after bonding layers together. It is not intended that thechannel be limited to the dimensions of the lane or channel. Forexample, in embodiments the width is between 1 and 5 mm, with the lengthbetween 5 and 100 mm; where the lane or channel has depth, it is between10-300 microns, (e.g., between 10 and 150 microns deep). The dimensionscan be adjusted based on the size of the reaction chamber and desiredarea to be imaged. For example, in embodiments the size of the reactionchamber or flow cell is dimensioned so that the area to be imaged isless than the size of a standard microscope slide (i.e. less than 75mm×25 mm) or considerably less (e.g., 35 mm×2.5 mm, or smaller).

In an embodiment, at least one channel has a cross sectional shape of acircle, rectangle, oval, or any other shape. Preferably, the flow rates,fluid viscosities, compositions, and geometries and sizes of the channelare selected so that fluid flow is laminar. Guidance for making suchdesign choices is readily available publicly available resources, forexample Acheson, Elementary Fluid Dynamics (Clarendon Press, 1990), andfrom software for modeling fluidics systems, e.g. SolidWorks fromDassault Systems. In an embodiment, at least one channel has passagecross-sections in the range of tens of square microns to a few squaremillimeters (e.g., maximal cross-sectional dimensions of from about 500μm to about 0.1 μm). In an embodiment, the flow rates in the range offrom a few nL/sec to a hundreds of μL/sec. In an embodiment, volumecapacities in are the range of from 1 μm to a few nL, e.g. 10-100 nL.

As used herein, the term “substrate” refers to a solid support material.The substrate can be non-porous or porous. The substrate can be rigid orflexible. A nonporous substrate generally provides a seal against bulkflow of liquids or gases. Exemplary solid supports include, but are notlimited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefin copolymers, polyimides etc.), nylon, ceramics,resins, Zeonor, silica or silica-based materials including silicon andmodified silicon, carbon, metals, inorganic glasses, optical fiberbundles, photopatternable dry film resists, UV-cured adhesives andpolymers. Particularly useful solid supports for some embodiments haveat least one surface located within a flow cell. The term “surface” isintended to mean an external part or external layer of a substrate. Thesurface can be in contact with another material such as a gas, liquid,gel, polymer, organic polymer, second surface of a similar or differentmaterial, metal, or coat. The surface, or regions thereof, can besubstantially flat. The substrate and/or the surface can have surfacefeatures such as wells, pits, channels, ridges, raised regions, pegs,posts or the like. The term “well” refers to a discrete concave featurein a substrate having a surface opening that is completely surrounded byinterstitial region(s) of the surface. Wells can have any of a varietyof shapes at their opening in a surface including but not limited toround, elliptical, square, polygonal, or star shaped (i.e., star shapedwith any number of vertices). The cross section of a well takenorthogonally with the surface may be curved, square, polygonal,hyperbolic, conical, or angular.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly indicates otherwise, between the upper and lowerlimit of that range, and any other stated or unstated intervening valuein, or smaller range of values within, that stated range is encompassedwithin the invention. The upper and lower limits of any such smallerrange (within a more broadly recited range) may independently beincluded in the smaller ranges, or as particular values themselves, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

As used herein, the term “raised handle” refers to the appendage 120that is elevated relative to the bottom of the frame 150. For example,when the frame 150 is in contact with a work surface (e.g., a tablesurface), the raised handle may be about 15 mm to about 25 mm from thesurface. In embodiments, the raised handle is about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 cm from the surface (for example when measured from theuppermost point or edge of the handle). In embodiments, the raisedhandle is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 cm from thesurface (for example when measured from the uppermost point or edge ofthe handle). In embodiments, the frame 150 is about 22 mm from thesurface. The raised handle is operatively attached to the flow cellcarrier so the user can grasp the flow cell carrier. In embodiments, theraised handle 120 does not make contact with the surface (aside from theattached frame 150 contact with the surface). A raised handle may beconsidered an ergonomic handle.

As used herein, the term “ergonomic handle” refers to an appendage 120that is designed to improve efficiency, comfort, or safety. For example,an ergonomic handle may be designed such that a user can align theirfingers on the handle in a manner that maximizes hand capacity and doesnot require wrist flexion, extension, or deviation, in order to allowthe user to maintain a neutral wrist posture. The ergonomic handle mayinclude additional features such as ridges, or other textures such asgrooves, indentations, rippling, stippling, or the like, to improvegrip. Alternatively, the ergonomic handle may further include a polymeror rubber coating (e.g., synthetic polymer, thermoplastic, or plastisolcoating). The polymer or rubber coating may provide a flexible, non-slipcushion to further promote the ergonomic design of the handle.

The term “injection molded” is used in accordance with its ordinarymeaning in the art and refers to a manufacturing process for producingparts by injecting hot (e.g., molten) material into a mold. Injectionmolding may be performed with a variety of input materials, such asmetals, glasses, elastomers, confections, and polymers (e.g.,thermoplastic and thermosetting polymers).

As used herein, the terms “sequencing”, “sequence determination”,“determining a nucleotide sequence”, and the like include determinationof a partial or complete sequence information (e.g., a sequence) of apolynucleotide being sequenced, and particularly physical processes forgenerating such sequence information. That is, the term includessequence comparisons, consensus sequence determination, contig assembly,fingerprinting, and like levels of information about a targetpolynucleotide, as well as the express identification and ordering ofnucleotides in a target polynucleotide. The term also includes thedetermination of the identification, ordering, and locations of one,two, or three of the four types of nucleotides within a targetpolynucleotide. In some embodiments, a sequencing process describedherein comprises contacting a template and an annealed primer with asuitable polymerase under conditions suitable for polymerase extensionand/or sequencing. The sequencing methods are preferably carried outwith the target polynucleotide arrayed on a solid substrate within aflow cell (i.e., within a channel of the flow cell). In an embodiment,the sequencing is sequencing by synthesis (SBS). Briefly, SBS methodsinvolve contacting target nucleic acids with one or more labelednucleotides (e.g., fluorescently labeled) in the presence of a DNApolymerase. Optionally, the labeled nucleotides can further include areversible termination property that terminates extension once thenucleotide has been incorporated. Thus, for embodiments that usereversible termination, a cleaving solution can be delivered to the flowcell (before or after detection occurs). Washes can be carried outbetween the various delivery steps. The cycle can then be repeated ntimes to extend the primer by n nucleotides, thereby detecting asequence of length n. Exemplary SBS procedures and detection platformsthat can be readily adapted for use with the methods of the presentdisclosure are described, for example, in Bentley et al., Nature456:53-59 (2008), WO 2004/018497; and WO 2007/123744, each of which isincorporated herein by reference in its entirety. In an embodiment,sequencing is pH-based DNA sequencing. The concept of pH-based DNAsequencing, has been described in the literature, including thefollowing references that are incorporated by reference: US2009/0026082;and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006) whichare incorporated herein by reference in their entirety. Other sequencingprocedures that use cyclic reactions can be used, such aspyrosequencing. Sequencing-by-ligation reactions are also usefulincluding, for example, those described in Shendure et al. Science309:1728-1732 (2005).

A nucleic acid can be amplified by a suitable method. The term“amplified” and “amplification” as used herein refers to subjecting atarget nucleic acid in a sample to a process that linearly orexponentially generates amplicon nucleic acids having the same orsubstantially the same (e.g., substantially identical) nucleotidesequence as the target nucleic acid, or segment thereof, and/or acomplement thereof. In some embodiments an amplification reactioncomprises a suitable thermal stable polymerase. Thermal stablepolymerases are known in the art and are stable for prolonged periods oftime, at temperature greater than 80° C. when compared to commonpolymerases found in most mammals. In certain embodiments the term“amplified” refers to a method that comprises a polymerase chainreaction (PCR). Conditions conducive to amplification (i.e.,amplification conditions) are well known and often comprise at least asuitable polymerase, a suitable template, a suitable primer or set ofprimers, suitable nucleotides (e.g., dNTPs), a suitable buffer, andapplication of suitable annealing, hybridization and/or extension timesand temperatures.

As used herein, the term “sequencing solution” is used in accordancewith its plain and ordinary meaning and refers to an aqueous mixturethat contains the reagents necessary to allow a dNTP or dNTP analogue toadd a nucleotide to a DNA strand by a DNA polymerase. In embodiments,the sequencing reaction mixture includes a buffer. In embodiments, thebuffer includes an acetate buffer, 3-(N-morpholino)propanesulfonic acid(MOPS) buffer, N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffer,phosphate-buffered saline (PBS) buffer,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer,N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO) buffer, borate buffer (e.g., borate buffered saline, sodiumborate buffer, boric acid buffer), 2-Amino-2-methyl-1,3-propanediol(AMPD) buffer, N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid(CAPSO) buffer, 2-Amino-2-methyl-1-propanol (AMP) buffer,4-(Cyclohexylamino)-1-butanesulfonic acid (CABS) buffer, glycine-NaOHbuffer, N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer,tris(hydroxymethyl)aminomethane (Tris) buffer, or aN-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer. In embodiments,the buffer is a borate buffer. In embodiments, the buffer is a CHESbuffer. In embodiments, the sequencing reaction mixture includesnucleotides, wherein the nucleotides include a reversible terminatingmoiety and a label covalently linked to the nucleotide via a cleavablelinker. In embodiments, the sequencing reaction mixture includes abuffer, DNA polymerase, detergent (e.g., Triton X), a chelator (e.g.,EDTA), and/or salts (e.g., ammonium sulfate, magnesium chloride, sodiumchloride, or potassium chloride).

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers), flow cells, flow cell carriers, and/orsupporting materials (e.g., packaging, buffers, written instructions forperforming a method, etc.) from one location to another. For example,kits include one or more enclosures (e.g., boxes) containing therelevant reaction reagents and/or supporting materials. As used herein,the term “fragmented kit” refers to a delivery system comprising two ormore separate containers that each contain a subportion of the total kitcomponents. The containers may be delivered to the intended recipienttogether or separately. For example, a first container may contain anenzyme for use in an assay, while a second container containsoligonucleotides. In contrast, a “combined kit” refers to a deliverysystem containing all of the components of a reaction assay in a singlecontainer (e.g., in a single box housing each of the desiredcomponents). The term “kit” includes both fragmented and combined kits.

The term “nucleic acid sequencing device” means an integrated system ofone or more chambers, ports, and channels that are interconnected and influid communication and designed for carrying out an analytical reactionor process, either alone or in cooperation with an appliance orinstrument that provides support functions, such as sample introduction,fluid and/or reagent driving means, temperature control, detectionsystems, data collection and/or integration systems, for the purpose ofdetermining the nucleic acid sequence of a template polynucleotide.Nucleic acid sequencing devices may further include valves, pumps, andspecialized functional coatings on interior walls. Nucleic acidsequencing devices may include a receiving unit, or platen, that orientsthe flow cell such that a maximal surface area of the flow cell isavailable to be exposed to an optical lens. An example flow cellreceiving unit is described in co-pending U.S. Provisional PatentApplication No. 62/952,790, entitled “FLOW CELL RECEIVER AND METHODS OFUSE” and having attorney docket number 051385-522P01US, which isincorporated herein by reference in its entirety. Other nucleic acidsequencing devices include those provided by Illumina™, Inc. (e.g.HiSeg™, MiSeg™, NextSeq™, or NovaSeq™ systems), Life Technologies™ (e.g.ABI PRISM™, or SOLiD™ systems), Pacific Biosciences (e.g. systems usingSMRT™ Technology such as the Sequel™ or RS II™ systems), or Qiagen (e.g.Genereader™ system).

The term “platen” is used in accordance with its plain ordinary meaningand refers to a flat platform. The platform composition may include asubstantially rigid material, for example, but not limited to, polymers,metals, inorganic oxide materials, such as glasses and sapphire-basedmaterials, and ceramics. In embodiments, the platen includes a surfacecoating. Numerous surface coatings are possible, such as a polymer thinfilm, where the polymer may be selected from a range of physical andsurface chemistry properties, such as, for example polyhalohydrocarbon,polystyrene, polyamide, polyimide and the like. Alternatively, a surfacecoating could be an inorganic coating, such as a silicon nitride,silicon carbide, silicon oxide, or diamond. In embodiments, a platen isa substantially planar platform.

The term “align” or “alignment” is used in accordance with its ordinarymeaning and refers to perfect alignment and alignment with relativelysmall, insignificant amount of deviation/misalignment (e.g., ≤5%).

The terms “fluid communication” or “fluidically coupled” refers to twospatial regions being connected together such that a liquid or gas mayflow between the two spatial regions.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

REFERENCES

Eltoukhy et al. U.S. Pat. No. 9,937,497

Reed et al. U.S. Pat. No. 8,951,781

What is claimed is:
 1. A flow cell carrier comprising: a flow cell; anda frame, the frame comprising: a handle; wherein the frame is configuredto retain the flow cell.
 2. The flow cell carrier of claim 1, whereinthe frame further comprises a pocket, the pocket comprising at least onebiasing feature.
 3. The flow cell carrier of claim 2, wherein the frameis configured to retain the flow cell within the pocket.
 4. The flowcell carrier of claim 3, wherein the frame is configured to retain theflow cell within the pocket such that a maximal surface area of the flowcell is available to be exposed to an optical lens.
 5. The flow cellcarrier of claim 1, wherein the retaining of the flow cell within thepocket further comprises constraining a first, a second, a third, afourth, a fifth, and a sixth degree of freedom of the flow cell.
 6. Theflow cell carrier of claim 1, wherein the frame is an injection moldedframe.
 7. The flow cell carrier of claim 1, wherein the handle is araised handle.
 8. The flow cell carrier of claim 1, wherein the frame isfurther configured to provide a gap between a work surface and the flowcell.
 9. The flow cell carrier of claim 1, wherein the frame furthercomprises at least one ferromagnetic pin.
 10. The flow cell carrier ofclaim 2, wherein the at least one biasing feature is a spring finger.11. The flow cell carrier of claim 2, wherein the at least one biasingfeature is a tab.
 12. The flow cell carrier of claim 1, furthercomprising a microchip.
 13. The flow cell carrier of claim 12, whereinthe microchip is an electronically erasable programmable read onlymemory (EEPROM) chip.
 14. A method of manufacturing a flow cell carrier,the method comprising: forming a frame, such that the frame comprises: ahandle, and securing a flow cell within the frame.
 15. A method ofsequencing a nucleic acid comprising: providing the flow cell carrier ofclaim 1, wherein the flow cell comprises a flow channel and isconfigured to serve as a reaction vessel in a nucleic acid sequencingdevice, positioning the flow cell carrier on the receiver of a nucleicacid sequencing device wherein the flow cell is exposed to an opticallens; conducting a plurality of sequencing events and detecting thesequencing events using the optical lens, thereby sequencing a nucleicacid.
 16. The method of claim 15, wherein the conducting of a pluralityof sequencing events comprises flowing a first aqueous solutioncomprising one or more reagents and one or more enzymes for DNAamplification through the flow channel.
 17. The method of claim 16,wherein the conducting of a plurality of sequencing events furthercomprises one or more wash cycles.
 18. The method of claim 17, whereinthe conducting of a plurality of sequencing events further comprisesflowing a sequencing solution through the flow channel that includes oneor more reagents and one or more enzymes for DNA sequencing.
 19. A kitfor sequencing a nucleic acid comprising: the flow cell carrier of claim1, wherein the flow cell is configured to serve as a reaction vessel ina nucleic acid sequencing device; a sequencing solution comprising oneor more reagents and one or more enzymes for DNA sequencing.
 20. The kitof claim 19, further comprising an aqueous solution comprising one ormore reagents and one or more enzymes for DNA amplification.